Self and microbial glycosylceramides perform important immunological functions as NKT cell antigens presented by CD1d, a lipid-binding MHC-like molecule. These functions range from host defense against pathogens to the regulation of cancer rejection, autoimmunity and allergy. Further, synthetic glycosylceramides can be used as adjuvants of vaccines and can modulate, through the release of either Thl or Th2 cytokines, various disease conditions including diabetes, experimental allergic encephalomyelitis, lupus and cancer rejection. Other glycosylceramides elicit adaptive T cell responses that may be involved in Multiple Sclerosis. Thus, the program project uses glycosylceramides as a model antigen family to study glycolipid antigen presentation by CD1d molecules, with potential clinical applications, as well as fundamental issues in lipid transport and recognition. A multidisciplinary approach encompassing glycolipid analytic and synthetic chemistry, protein expression, biophysics and structural biology, as well as cellular immunology and cell biology, has been assembled to identify NKT ligands from natural sources and study fundamental aspects of their immunobiology. The past funding period has produced landmark accomplishments in identifying key self and microbial NKT cell antigens, characterizing lipid transfer proteins essential for their presentation, elucidating their crystal structures and developing a panel of synthetic variants to study their cell biology and their functional impact in vaccine and immunomodulation. These approaches will be pursued with the goal of developing a basic understanding of the cell biology of lipid uptake, trafficking and loading, of the biophysical and structural aspects of their binding to protein receptors such as CD1d, TCR and other lipid binding proteins; and integrating this knowledge in vivo at the functional level during NKT cell mediated immune responses to infection or to synthetic adjuvants. The individual research projects are as follows. Project 1. Savage: Identification and synthesis of microbial NKT ligands and design of structural variants and conjugates to understand their functional properties. Project 2. Teyton: Biochemical, biophysical and structural aspects of lipid interaction with CD1d, TCR and other lipid binding or transfer protein. Project 3. Bendelac: Functions associated with NKT ligand recognition in vivo in mice and intracellular trafficking properties of glycosylceramides